The interaction between a host macrophage and infecting bacterium is central to TB pathogenesis. Once Mtb is internalized, the phagosome is the point of interface between macrophage and bacterium, yet we understand little of how the phagosome evolves in the course of Mtb infection and how those changes might shift the host/pathogen balance toward or away from control. Comprehensive proteomic profiling would offer a window into phagosome composition over the course of infection and the differences in composition between phagosomes in macrophages that are able to control infection and those that permit Mtb growth. To date, targeted studies of individual proteins on the Mtb-containing phagosomal membrane have identified a few correlates of phagosome state. However, systematic, comprehensive proteomic studies of the Mtb-containing phagosome have been limited. In part, this limitation has arisen from available technical approaches. Most studies of Mtb-containing phagosomes have relied on purification of this subcellular fraction. Achieving purity of this fraction is challenging at best, and is more difficult within the constraints of technologies available in a BSL3 setting. Beads containing purified Mtb products have additionally been used to isolate the phagosomal fraction, but how well individual bacterial products represent the intact Mtb surface is not clear. To fill gaps in our understanding of phagosome composition and evolution in Mtb infection of macrophages, we propose to build upon recent advances in protein engineering and comparative proteomics using an approach successfully applied to other biological questions: proximity labeling using the promiscuous, secreted biotin ligase TurboID. We will first optimize this system for use in Mtb; we will then apply it to profile the composition of the Mtb-containing phagosome under conditions that promote or fail to promote control of infection. In Aim 1, we will systematically test strategies for secretion and surface localization of TurboID to engineer an Mtb strain optimal for proximity ligation experiments. In Aim 2, we will optimize analytical approaches to proteome profiling with our engineered strain. Following optimization, we will profile and compare phagosomal composition in macrophages treated with stimuli that promote varying degrees of control of Mtb. In Aim 3, we will use this system to profile and compare phagosomal composition from macrophages infected with wild-type Mtb or two mutants that lack virulence factors that interact with the phagosomal membrane. Upon achieving this work, we anticipate having developed and validated a system for profiling the composition of the Mtb- containing phagosome with high temporal and spatial resolution. We expect to have identified key differences between phagosomes that control or fail to control infection. We anticipate that our results will fuel future mechanistic studies of the contribution of individual phagosomal factors to control of infection. Further, we anticipate these results will ultimately inform future host-directed anti-TB therapeutics that enhance host control of infection.